1. Field of the Invention
The present invention relates to a double-resonance-absorption microscope that achieves super-resolution by using a double-resonance absorption process.
2. Description of the Related Art
In recent years, there have been developed various types of optical microscopes with high performances and multiple functions, along with the developments in the peripheral technologies including laser and electronic graphics technologies. As one of these optical microscopes, there has been proposed, by the inventor of the present invention, a microscope capable of contrast control of an image and chemical analysis of a sample by the use of a double resonance absorption process caused by irradiating the sample with plural wavelengths of light. This is hereinafter referred to as a double-resonance-absorption microscope (see Japanese patent application No. 329165/1994).
This double-resonance-absorption microscope can, by using the double resonance absorption process, select a specific kind of molecule and observe absorption and fluorescence caused by a specific optical transition. The principle is described below. Sample molecules forming a sample in a ground state (state S0 of FIG. 1) have electrons in a valence electron orbit. These electrons in the valence orbit are first excited to a first electronic excited state (abbreviated state S1 in FIG. 1) by light, such as a laser beam, of a resonance wavelength λ1, and are subsequently excited to a second electronic excited state or a higher excited state (abbreviated state S2 in FIG. 3) by light of a resonance wavelength λ2. Then, the molecules in this excited state return to the ground state while emitting a fluorescence or a phosphorescence as illustrated in FIG. 4. An absorption image or a luminous image is observed by using the absorption process of FIG. 2 or the emission of the fluorescence or phosphorescence illustrated in FIG. 4.
In the excitation process to state S1, the number of molecules in state S1 in a unit volume increases as the intensity of the irradiating light increases. Since a linear absorption coefficient is given as the product of an absorption cross-section per molecule and the number of molecules per unit volume in the excitation process to state S2, the linear absorption coefficient for the resonance wavelength λ2 subsequently irradiated depends on the intensity of the light of the resonance wavelength λ1 first irradiated. Accordingly, the linear absorption coefficient for the resonance wavelength λ2 (hereinafter often simply referred to as the wavelength λ2) can be controlled with the intensity of the light of the resonance wavelength λ1 (hereinafter often simply referred to as the wavelength λ1). This indicates that, when irradiating a sample with two wavelengths λ1 and λ2 of light and observing a transmission image obtained by the wavelength λ2, contrast of the transmission image can be completely controlled with the light of the wavelength λ1. Further, when the excited molecules deexcitate from state S2 by emitting fluorescence of phosphorescence, its luminous intensity is proportional to the number of molecules in state S2. Therefore, the image contrast can be controlled where the instrument is used as a fluorescence microscope.
Furthermore, the double-resonance-absorption microscope enables chemical analysis, as well as contrast control. Since the outermost valance orbit in FIG. 1 has an energy level intrinsic to each individual sample molecule, the wavelength λ1 differs among each individual sample molecule. Also, the wavelength λ2 is also intrinsic to each individual sample molecule. The prior art microscope which performs its irradiation and observation with a single wavelength can observe an absorption image or a fluorescence image of a specific molecule to a certain extent. However, it cannot accurately identify the chemical composition of a sample because, in general, the ranges of absorption wavelengths of some molecules overlap with each other. In contrast, the double-resonance-absorption microscope can limit absorbing or emitting molecules by the two wavelengths of λ1 and λ2 and thus identify the chemical composition of the sample more precisely than the prior art instrument.
Moreover, when the valance electron is to be excited, only light having a certain electric-field vector with respect to the molecular axis is intensively absorbed. Thus, if an absorption image or fluorescence image is obtained while determining the polarization directions of the wavelengths λ1 and λ2, the orientation directions can also be identified for the same molecule.
There has also been proposed, by the inventor of the present invention, another double-resonance-absorption microscope of a high spatial resolution exceeding the diffraction limit by using the double resonance absorption process. In the double resonance absorption process, there exist some molecules which emit extremely weak fluorescence from state S2 as in FIG. 5. The molecules having such optical properties experience a unique phenomenon as described below.
FIG. 6 is a conceptual diagram of a double resonance absorption process similarly to FIG. 5. The x-axis is set along the horizontal axis of FIG. 6 to express the spread of spatial distance. In FIG. 6, a spatial area A1 is irradiated with both wavelengths λ1 and λ2 of light, while a spatial area A0 is irradiated with only the wavelength λ1 of light. These spatial areas A1 and A2 are referred to as the fluorescence inhibited area and the fluorescence area, respectively.
In the spatial area A0, a great number of molecules in state S1 are generated by the excitation with the wavelength λ1, and fluorescence emitted with a wavelength λ3 can be observed. In the spatial area A1, however, the molecules in state S1 are instantly excited to the higher state S2 by irradiation with the wavelength λ2 and thus disappear. As a result, the fluorescence is completely inhibited in the spatial area A1 because the fluorescence of the wavelength λ3 will not be emitted at all and because the fluorescence from molecules in state S2 does not exist intrinsically. Consequently, the fluorescence is emitted only from the spatial area A0. Such a phenomenon has been observed with some kinds of molecules.
Accordingly, in the prior art scanning laser microscope or the like, the size of a microbeam that is created on an observed sample by focusing laser light is determined by the diffraction limit that depends on the numerical aperture of the focusing lens and on the wavelength. It cannot be theoretically expected, therefore, that higher spatial resolution will be obtained. In the phenomenon illustrated in FIG. 6, light of wavelength λ1 and light of wavelength λ2 are made to overlap with each other spatially and thus fluorescence region is restricted with illumination of light of wavelength λ2. Therefore, if we take notice of a region irradiated with light of wavelength λ1, the fluorescence region is narrower than the size of the beam that is determined by the numerical aperture of the focusing lens and by the wavelength. This substantially improves the spatial resolution. The present inventor's double-resonance-absorption microscope (see: Japanese patent application No. 302232/1996) uses this principle to achieve a microscope having super-resolution exceeding the diffraction limit.
In an attempt to further enhance the super-resolution of the double-resonance-absorption microscope, the present inventor has already made a proposal for adjusting the sample to make full use of the functions and for timing at which light of wavelength λ1 and light of wavelength λ2 are directed to the sample (see Japanese patent application No. 255444/1997). In particular, the sample is stained with staining molecules, which have at least three quantum states S0, S1, and S2 including the ground state. Furthermore, when these molecules are deexciting from a higher quantum state excluding state S1 to the ground state, a thermal relaxation process is more prevalent than a relaxation process due to fluorescence. These molecules are hereinafter referred to as the fluorescence labeler molecules. In a sample, such fluorescence labeler molecules and biological molecules biologically stained are chemically bonded. This sample is irradiated with light of wavelength λ1 to promote the fluorescence labeler molecules to state S1. Immediately thereafter, the sample is irradiated with light of wavelength λ2 to excite the fluorescence labeler molecules to a still higher quantum level. Consequently, fluorescence from state S1 can be effectively suppressed. At this time, the aforementioned artificial spatial suppression of the fluorescence region is performed. In this way, a further improvement of the spatial resolution can be accomplished.
The optical properties of the above-described fluorescence labeler molecules can be explained from a quantum-chemical point of view as follows. Generally, molecules are bonded by a σ bond or π bond of atoms constituting the molecules. In other words, molecular orbits have σ-molecular orbits or π-molecular orbits. Electrons existing in these molecular orbits play a key role in bonding together atoms. Among them, electrons in σ-molecular orbits strongly bond atoms and determine the intermolecular distances within each molecule (i.e., the skeleton of the molecule). On the other hand, electrons in π-molecular orbits contribute little to bonding of atoms. Rather, they are bound to the whole molecule with a quite weak force.
Where electrons existing in σ-molecular orbits are excited with light, interatomic spaces in a molecule often vary greatly, resulting in a large structural change including dissociation of the molecule. As a result, the kinetic energy of the atoms or the energy given to the molecule by the light to cause the structural change is almost fully changed into thermal energy. Therefore, excitation energy is not consumed in the form of light, i.e., fluorescence. Since a molecular structural change takes place quite quickly (e.g., in a time shorter than picosecond), if fluorescence occurs during the process, the life of the fluorescent light is quite short on the other hand, where electrons in π-molecular orbits are excited, the molecular structure itself is hardly varied. The electrons stay in higher-order discrete quantum levels for a long time. They release fluorescent light for orders of nanoseconds and deexcite.
In quantum chemistry, having a π-molecular orbit is equivalent to having a double bond for a molecule. It is necessary that a fluorescence labeler molecule having a rich amount of double bonds be selected. It has been confirmed that among molecules having double bonds, six-membered ring molecules such as benzene and belladine show quite weak fluorescence from state S2 (e.g., M. Fujii et. al., Chem. Phys. Lett. 171 (1990) 341). Therefore, if molecules including six-membered rings such as benzene and belladine are selected as fluorescence labeler molecules, the life of fluorescence from molecules in state S1 is prolonged. In addition, fluorescence from molecules can be easily suppressed by exciting them from state S1 to state S2 by light illumination. Hence, it is possible to make effective use of the super-resolution of the aforementioned double-resonance absorption microscope.
That is, if a sample is stained with these fluorescence labeler molecules and an observation is made, a fluorescence image with high spatial resolution can be obtained. Additionally, only a desired chemical structure of a biological sample can be stained by adjusting the chemical groups on side chains of the fluorescence labeler molecules. In consequence, even detailed chemical compositions of the sample can be analyzed.
Generally, a double-resonance-absorption process takes place only when two wavelengths of light, state of polarization, and other factors satisfy certain conditions. Therefore, use of this process makes it possible to know the molecular structure quite accurately. In particular, the direction of polarization of light has a strong correlation with the direction of orientation of the molecules. A strong double-resonance-absorption process occurs when the directions of polarization of two wavelengths of light have certain angles with respect to the direction of orientation of the molecules. Accordingly, the degree of extinction of fluorescence is varied by illuminating the sample with the two wavelengths of light and rotating their directions of polarization. Hence, information about the spatial orientation of a structure to be observed can be obtained by observing the manner in which the extinction varies. This can also be made possible by adjusting the two wavelengths of light.
Another method as proposed in Japanese patent application No. 255444/1997 improves the S/N of the resulting fluorescence image and suppresses the fluorescence more effectively by appropriately adjusting the timing at which the wavelengths λ1 and λ2 of light are illuminated.
In addition, the present inventor has proposed a method of improving the S/N and suppression of fluorescence further by more ingeniously devising the timing at which the wavelengths λ1 and λ2 of light are illuminated (see Japanese patent application No. 97924/1998).
The region irradiated with the light of wavelength λ1 overlaps a part of the region irradiated with the light of wavelength λ2 as mentioned previously. This can be accomplished by shaping the light of wavelength λ2 into a hollow beam (i.e., having a central portion (around the axis) of zero intensity and having an intensity distribution symmetrical with respect to the axis), bringing this hollow beam into registry with a part of the light of wavelength λ1, and focusing the light onto a sample. FIG. 7 is a conceptual diagram illustrating this overlap and the fluorescence suppression caused thereby. The light of wavelength λ2 is shaped into a hollow beam by a phase plate as shown in FIG. 8. The light of wavelength λ2 in the form of a hollow beam and light of wavelength λ1 are made to overlap with each other. This suppresses fluorescence other than in a region close to the optical axis where the intensity of light of wavelength λ2 is zero. Only fluorescence is observed which arises from the fluorescence labeler molecules (or sample molecules) existing in a region narrower than the spread of the light of wavelength λ1. As a result, super-resolution is developed.
The phase plate of FIG. 8 gives a phase difference of π to the light of wavelength λ2 with respect to the optical axis. The light of wavelength λ2 is passed through this phase plate, inverting the phase of the light of wavelength λ2 in the region on and close to the optical axis. As a consequence, the electric field strength in the region close to the optical axis is brought to zero. Thus, the light of wavelength λ2, which assumes the form of a hollow beam, can be obtained.
While the double-resonance-absorption microscope developed thus far by the present inventor exhibits excellent super-resolution and analyzing capability (which provide great usefulness) and technical superiority, the actual situation is that this instrument still has points to be improved as described below. Light for exciting sample molecules (i.e., molecules constituting a sample) from state S0 to S1 is hereinafter referred to as “pump light”. Light for exciting molecules in state S1 to state S2 is referred to as “erase light”. Erase light assuming the form of a hollow beam is referred to as “hollow erase light”. Excitation from state S0 to state S1 is abbreviated as “excitation S0→S1”. Excitation from state S1 to state S2 is abbreviated as “excitation S1→S2”. Where a sample is stained with fluorescence labeler molecules to realize super-resolution more effectively, the sample molecules are none other than the fluorescence labeler molecules.
[I] Ideal Hollow Erase Light
First, in order to realize super-resolution by a double-resonance-absorption microscope as expected theoretically by suppression of a fluorescence region due to partial overlap between regions irradiated with pump light and erase light, respectively, it is necessary that the hollow erase light assume the anticipated form of a hollow beam. Disturbance of this shape of the hollow beam, i.e., disturbance of the intensity distribution, leads to deterioration of the microscope image.
Lasers are often used as light sources for erase light. In order to shape erase light from a light source into the expected hollow beam, it is absolutely necessary that the beam profile of the laser light be regulated. That is, the intensity distribution of the beam must be symmetrical with respect to the optical axis. However, the beam profile of adyelaser, for example, is close to a triangle. Furthermore, the intensity distribution it not uniform. Therefore, it may be difficult to obtain the expected hollow beam. Consequently, the beam profile focused onto a sample shows a disturbed beam pattern. This causes a deterioration of the resolution or image quality of the microscope.
Furthermore, it has been proposed to obtain hollow erase light via a zonal aperture. However, if this aperture is utilized, it is difficult to perform alignment or focusing. Along adjusting time and a very large amount of labor are required until a good image is obtained. Moreover, skillfulness for this is necessary. These are unfavorable in practical applications.
Additionally, a first-order Bessel beam having an ideal beam profile as hollow erase light has been proposed. As shown in FIG. 9, if the first-order Bessel beam is caused to make one revolution around the optical axis, the phase varies by 2π. Theoretically, two points that are symmetrical with respect to the optical axis are shifted in phase with respect to each other by π. Therefore, on the axis, the electric fields completely cancel out, and the strength of the resultant field is zero. In practice, however, the phase plane of the laser light is not completely uniform within the plane of the beam. As it goes away from the center of the beam, the phase plane becomes more disturbed. Therefore, when a first-order Bessel beam is created, cancellation of the electric fields becomes incomplete due to the disturbance of the phase plane. In the resulting first-order Bessel beam, the intensity at the center of the beam is not exactly zero. It cannot be said that this hollow erase light is ideal for a double-resonance-absorption microscope.
Accordingly, there is a demand for a technique capable of generating both super-resolution and ideal hollow erase light.
[II] Operability and Maintainability of Light Source
Secondly, the double-resonance-absorption microscope is, of course, required to have good operability and maintainability in the same way as other microscopes. The above-described double-resonance-absorption microscope uses a wavelength variable laser such as a dye laser or an optical parametric oscillator (OPO) as a light source and so this technique can be applied to resonance conditions of various fluorescence labeler molecules. However, conventional dye lasers suffer from drops in the amount of light due to deterioration of dyes. Also, frequent and cumbersome operations for replacing the dye are necessary. Therefore, the dye lasers are not favorable from a practical point of view. The OPO is convenient but is a quite accurate optical system. Therefore, humidity and temperature must be strictly controlled. Furthermore, the used nonlinear optical crystal has a short life and is expensive. Since the whole system is also expensive, a heavy burden is imposed on the user in servicing the light source. Accordingly, there is a demand for a light source that has excellent operability and maintainability.
[III] Mixing of Excitation Light into Detection Signal
Thirdly, depending on the molecules to be excited, the wavelength range of fluorescence from the molecules maybe close to or overlap with the wavelengths of erase light and pump light for exciting the molecules. Therefore, when the resulting fluorescence signal is detected, the excitation light forms background light. This may make it difficult to extract the fluorescence signal to be measured. Especially, for the erase light, it is necessary to excite the molecules from state S1 to state S2 and so the intensity is relatively high. Its effects need to be taken into consideration. For example, in the above-cited technique (Japanese patent application No. 97924/1998), where a sample is stained with fluorescence labeler molecules, if rhodamine 6G is used as the fluorescence labeler molecules, the fluorescence region extends from a wavelength of about 530 nm to a wavelength of 650 nm as shown in FIG. 10. The wavelength of the pump light for rhodamine 6G is 532 nm, while the wavelength of the erase light is 599 nm. Therefore, the fluorescence region overlaps with the exciting wavelengths. Consequently, the S/N of the obtained fluorescence image is not good. Accordingly, there is a demand for a technique for suppressing mixing of the excitation light into the detected signal, thus achieving high S/N.
[IX] 3D Spatial Resolution
Fourthly, attempts have been made to improve the performance of microscopes in recent years. In this connection, realizing sufficient depth resolution in the direction of the optical axis, i.e., 3D (three-dimensional) spatial resolution is a great subject. However, the double-resonance-absorption microscope has no depth resolution in the direction of the optical axis, similar to other conventional optical microscopes. As mentioned previously, where the region irradiated with pump light is made to overlap with the region irradiated with erase light, the spatial resolution is improved only in two dimensions. In peripheral regions of the pump light beam with which the erase light overlaps, fluorescence is suppressed. However, on the optical axis in the hollow portion of the erase light, fluorescence is not suppressed at all, but rather molecules on the optical axis emit light. That is, in theory, there is no depth resolution in the direction of the optical axis. To have depth resolution, pinholes may be placed over the whole surface of the detector and at the confocal position. Unfortunately, this may present practical problems. That is, alignment of the focusing optical system including the pinholes is made complex. Furthermore, the number of photons of fluorescence reaching the detector is reduced. Therefore, if a microscope equipped with an uncomplex focusing optical system and having depth resolution, in addition to 2D (two-dimensional) resolution, can be designed, then a microscope having unprecedented high performance can be accomplished. Accordingly, there is a demand for a technique capable of realizing excellent 3D spatial resolution.
[X] Fluorescence Correlation Method
Fifthly, a fluorescence correlation method capable of making fluorescence analysis at a single molecular level has been known. Where a fluorescence analysis is performed by the prior art fluorescence correlation method using a double-resonance-absorption microscope, some problems remain to be solved. That is, where a pulsed light source such as a pulsed laser is used as a pulsed light source, it is quite difficult to precisely measure a fluorescence correlation function only depending on a fluorescent phenomenon. This problem will be described in further detail below. Accordingly, there is a demand for a novel fluorescence correlation method capable of precisely measuring a fluorescence correlation function relying only on a fluorescent phenomenon, even if a pulsed light source is used in a double-resonance-absorption microscope.